Bottom Line:
After 48 h, B. bacilliformis transitioned from bacillus-shape to a non-motile, small coccoid form and appeared to be digested along with the blood meal in both fly species.The capacity of L. longipalpis to transmit viable B. bacilliformis from infected to uninfected meals was analyzed via interrupted feeds.This study provides significant information toward understanding colonization of sand flies by B. bacilliformis and also demonstrates the utility of L. longipalpis as a user-friendly, live-vector model system for studying this severely neglected tropical disease.

Affiliation: Division of Biological Sciences, University of Montana, Missoula, Montana, United States of America.

ABSTRACTBartonella bacilliformis is a pathogenic bacterium transmitted to humans presumably by bites of phlebotomine sand flies, infection with which results in a bi-phasic syndrome termed Carrión's disease. After constructing a low-passage GFP-labeled strain of B. bacilliformis, we artificially infected Lutzomyia verrucarum and L. longipalpis populations, and subsequently monitored colonization of sand flies by fluorescence microscopy. Initially, colonization of the two fly species was indistinguishable, with bacteria exhibiting a high degree of motility, yet still confined to the abdominal midgut. After 48 h, B. bacilliformis transitioned from bacillus-shape to a non-motile, small coccoid form and appeared to be digested along with the blood meal in both fly species. Differences in colonization patterns became evident at 72 h when B. bacilliformis was observed at relatively high density outside the peritrophic membrane in the lumen of the midgut in L. verrucarum, but colonization of L. longipalpis was limited to the blood meal within the intra-peritrophic space of the abdominal midgut, and the majority of bacteria were digested along with the blood meal by day 7. The viability of B. bacilliformis in L. longipalpis was assessed by artificially infecting, homogenizing, and plating for determination of colony-forming units in individual flies over a 13-d time course. Bacteria remained viable at relatively high density for approximately seven days, suggesting that L. longipalpis could potentially serve as a vector. The capacity of L. longipalpis to transmit viable B. bacilliformis from infected to uninfected meals was analyzed via interrupted feeds. No viable bacteria were retrieved from uninfected blood meals in these experiments. This study provides significant information toward understanding colonization of sand flies by B. bacilliformis and also demonstrates the utility of L. longipalpis as a user-friendly, live-vector model system for studying this severely neglected tropical disease.

pntd.0004128.g003: B. bacilliformis colonization of sand flies 48h post-blood meal.At this time point, colonization of L. verrucarum (A-B) and L. longipalpis (C-D) was indistinguishable. Bacteria appeared to adhere to peritrophic membrane (PM) and although colonization was limited to abdominal midgut (shown here) on rare occasion, appeared outside the PM (arrow). The majority of bacteria changed morphology into non-motile small coccoid forms (seeS3 Video), and appeared more numerous than at 24h. Images acquired (1000x) with fluorescence (GFP) or phase contrast (PC) microscopy.

Mentions:
At 48h post-blood meal, bacteria appeared more numerous (as compared to the 24-h time point) and had changed their morphology to smaller coccoid forms of approximately 1 μm in diameter (Fig 3). On rare occasions, bacteria were observed on the apparent external side of the PM and one of the L. verrucarum sampled at this time point showed a small number of bacteria in the esophagus. In addition to changing shape, motility was significantly decreased and bacteria appeared adherent to the inner wall of the PM (S3 Video). At 48h, colonization of both fly species by B. bacilliformis appeared the same.

pntd.0004128.g003: B. bacilliformis colonization of sand flies 48h post-blood meal.At this time point, colonization of L. verrucarum (A-B) and L. longipalpis (C-D) was indistinguishable. Bacteria appeared to adhere to peritrophic membrane (PM) and although colonization was limited to abdominal midgut (shown here) on rare occasion, appeared outside the PM (arrow). The majority of bacteria changed morphology into non-motile small coccoid forms (seeS3 Video), and appeared more numerous than at 24h. Images acquired (1000x) with fluorescence (GFP) or phase contrast (PC) microscopy.

Mentions:
At 48h post-blood meal, bacteria appeared more numerous (as compared to the 24-h time point) and had changed their morphology to smaller coccoid forms of approximately 1 μm in diameter (Fig 3). On rare occasions, bacteria were observed on the apparent external side of the PM and one of the L. verrucarum sampled at this time point showed a small number of bacteria in the esophagus. In addition to changing shape, motility was significantly decreased and bacteria appeared adherent to the inner wall of the PM (S3 Video). At 48h, colonization of both fly species by B. bacilliformis appeared the same.

Bottom Line:
After 48 h, B. bacilliformis transitioned from bacillus-shape to a non-motile, small coccoid form and appeared to be digested along with the blood meal in both fly species.The capacity of L. longipalpis to transmit viable B. bacilliformis from infected to uninfected meals was analyzed via interrupted feeds.This study provides significant information toward understanding colonization of sand flies by B. bacilliformis and also demonstrates the utility of L. longipalpis as a user-friendly, live-vector model system for studying this severely neglected tropical disease.

Affiliation:
Division of Biological Sciences, University of Montana, Missoula, Montana, United States of America.

ABSTRACTBartonella bacilliformis is a pathogenic bacterium transmitted to humans presumably by bites of phlebotomine sand flies, infection with which results in a bi-phasic syndrome termed Carrión's disease. After constructing a low-passage GFP-labeled strain of B. bacilliformis, we artificially infected Lutzomyia verrucarum and L. longipalpis populations, and subsequently monitored colonization of sand flies by fluorescence microscopy. Initially, colonization of the two fly species was indistinguishable, with bacteria exhibiting a high degree of motility, yet still confined to the abdominal midgut. After 48 h, B. bacilliformis transitioned from bacillus-shape to a non-motile, small coccoid form and appeared to be digested along with the blood meal in both fly species. Differences in colonization patterns became evident at 72 h when B. bacilliformis was observed at relatively high density outside the peritrophic membrane in the lumen of the midgut in L. verrucarum, but colonization of L. longipalpis was limited to the blood meal within the intra-peritrophic space of the abdominal midgut, and the majority of bacteria were digested along with the blood meal by day 7. The viability of B. bacilliformis in L. longipalpis was assessed by artificially infecting, homogenizing, and plating for determination of colony-forming units in individual flies over a 13-d time course. Bacteria remained viable at relatively high density for approximately seven days, suggesting that L. longipalpis could potentially serve as a vector. The capacity of L. longipalpis to transmit viable B. bacilliformis from infected to uninfected meals was analyzed via interrupted feeds. No viable bacteria were retrieved from uninfected blood meals in these experiments. This study provides significant information toward understanding colonization of sand flies by B. bacilliformis and also demonstrates the utility of L. longipalpis as a user-friendly, live-vector model system for studying this severely neglected tropical disease.